We intend to use various variants of NOD mice to address the hypothesis that CD4+CD25+ T cells regulates IDDM and that a decrease in their function or number causes IDDM in older NOD female mice. We will ask whether iv or oral peptide administration induces anergy and up- regulates GRAIL expression (a recently identified anergy gene). We will additionally ask whether CD4+CD25+ T cells play a role in this peptide-induced anergy. Using linear amplification of mRNA and subsequent microarray technology, we will examine the genes expressed in CD4+CD25+ T cells), into conventional or immuno-deficient NOD mice reconstituted with diabetogenic T cells, we will analyze tolerance induction imposed by various routes of peptide administration in collaboration with Dr. Weiner. Three specific aims are proposed to examine these hypotheses. The novelty of the proposed studies arises from two recent experimental observations in my lab. We identified a gene, GRAIL, that functions to block the transcription of IL-2 mRNA in CD4+ T cells and whose expression in CD4+ T cells correlates with the phenotype of anergy. As determined by sequence motif searchers, this anergy gene encodes a trans-membrane protein, whose expression could function to serve as a reliable marker for the anergic (tolerant) state in CD4+ T cells. We postulate that GRAIL expression will provide a novel and effective screen for the anergic phenotype in mice and can possibly demonstrate in man in the studies proposed by Dr. Hafler. The second important observation was the elucidation of a mechanism of action of CD4+CD25+ T suppressor cells. In these studies, we demonstrated that CD4+CD25+ T cells act as classic suppressor cells when co-cultured with CD4+CD25- T cells following activation of both sub-populations. The intriguing finding was the up-regulation of GRAIL expression and the blockade of IL-2 mRNA and protein production in the suppressed CD4+CD25- T cells, anergy in the classic sense. More recent findings have suggested a role for GRAIL expression in peptide-induced tolerance in vivo in mice.